Encyclopedia
Please note this is a comparison between Version 1 by Ye Runle and Version 3 by Nora Tang.
Genetic engineering refers to the specific molecular biological modification of DNA sequences. With the rapid development of genetic engineering methods, especially the breakthroughs in guiding endonuclease technology, gene remodeling of crops has become simpler, more precise, and efficient. Genetic engineering techniques can be used to develop crops with superior traits such as high trace elements and high plant nutrients, providing an important tool to meet the needs of nearly 7.6 billion people in the world for crop yield and quality and to achieve sustainable development.
- genetic engineering
- transgenic technology
- gene editing technology
1. Transgenic Technology
Transgenic technology is a method that transmits a piece of DNA in line with the wishes of researchers to the target cell through physical, chemical, biological, or comprehensive methods and integrates it into the target cell genome to achieve its expression [1][4]. Since the birth of herbicide-resistant transgenic tobacco mediated by Agrobacterium tumefaciens in 1983 [2][5], the research of plant transgenic technology has been deepening constantly. Accordingly, a growing number of technologies have been developed, such as Agrobacterium tumefaciens transformation technology, virus-vector-mediated technology, biolistic technology, liposome-mediated transfection, ultrasound-mediated transformation, polyethylene glycol induction, pollen-tube pathway method, ovary injection, etc. [3][7].
The most commonly used transgenic techniques in plants are Agrobacterium tumefaciens transformation and biolistic technology. For the former, the T-DNA region and Vir region of the plasmid are of great significance. With the help of Vir-region-associated proteins and other Agrobacterium tumefaciens genes, T-DNA can be randomly inserted into the cell genome of injured plants and expressed [4][8]. Consequently, using this property, the target sequence can be inserted into the multi-clone site of Agrobacterium tumefaciens and integrated into the host cell genome with T-DNA at random. This method is simple and effective, yet obviously limited by the host range. Subsequently, the emergence of biolistic technology to some extent broke the restrictions of the host range [5][9], in which the third generation utilizes high-pressure inert gas as the driving force to insert gold or tungsten particles wrapped in the target DNA into the recipient cells to achieve transformation [5][6][9,10].
The feasibility of this method lies in that the particle size is suitably small (diameter of 0.6 μm) [5][9]. In addition, the driving force is also sufficiently strong, with impact pressures up to 900 psi [6][10]. However, it still has apparent defects, for instance, the efficiency of DNA integration into the genome is not high, and multiple particles entering simultaneously may easily lead to a higher number of DNA copies, resulting in more frequent gene inactivation or silencing [7][11]. In order to develop a technique with wider application and more stable transformation, Ribeiro et al. [6][10], using cotton hypocotyl as the explant, developed cotton with high resistance to Anthonomus grandis with the joint use of Agrobacterium tumefaciens transformation and biolistic technology, and this character was found to be stably inheritable. Gurusaravanan et al. [8][12], using cotton stem tip as the explant, transformed the uidA gene in cotton using Agrobacterium tumefaciens with the aid of microinjection and ultrasound, and the transformation efficiency was up to 20.25%. In addition, the strategy of creating transgenes based on nanoparticles has also been pursued by researchers owing to its comprehensive advantages such as excellent transformation efficiency, biocompatibility, and less harm to the host [9][13]. Although it is still in its infancy, its application potential has been proved in a variety of model plants [9][13].
Transgenic technology developed from traditional breeding technology; they are both essentially the genetic integration of target genes. All kinds of transgenic technologies possess obvious merits and demerits; thus, we tcan figure out the most suitable transformation method can be figured out bby focusing on the purpose of the research.
Generally speaking, transgenic technology breaks the species restrictions of conventional organisms, and its use can achieve crop improvement more purposefully and efficiently.
2. Gene Editing Technology
There are many types of gene editing tools, such as zinc-finger nucleases (ZFNs) [10][14], transcription-activator-like effector nucleases (TALENs) [11][15], and clustered regulatory interspaced short palindromic repeat (CRISPR)/CRISPR-associated 9 protein (Cas9) systems, that are based on biological cutting methods [12][16] and have broad potential in crop breeding and improvement.
The traditional gene editing process is mainly divided into two steps: First, the target sites of double-stranded DNA are cleaved by a nuclease system with some degree of engineering modification to produce double-stranded breaks (DSBs). Second, since DSBs are lethal to cells in many cases, to ensure genome integrity, the cells will initiate two endogenous repair pathways (non-homologous end junction (NHEJ) pathway and homologous recombination (HR) pathway) for repair.
Among them, NHEJ has high repair efficiency but poor stability, and it is easy to perform the insertion or deletion of small fragments in the repair site region [13][14][17,18]. In addition, in the presence of homologous templates, fractures can be repaired using the HR approach. This repair method is more accurate and at least two orders of magnitude more efficient than the traditional homologous recombination method used for gene shooting [10][13][14][14,17,18]. However, compared with NHEJ repair, HR repair efficiency is still lower [10][13][14][14,17,18]. To make crop improvement more flexible, it is necessary to optimize the efficiency of accurate repair, and researchers have made a series of efforts to improve the efficiency of HR repair. For example, in plants, Matthew et al. [15][19], in the study based on homologous recombination knock out of the rice chlorophyll a oxygenase gene (CAO1), used the Cpf1 nuclease to edit CAO1 and found that the homologous recombination repair efficiency was improved, with an efficiency of 8%. Wang et al. [16][20] found that, by increasing the number of homologous templates, endogenous actin 1 gene (ACT1) and glutathione S-transferase gene (GST) were transported by the wheat dwarf virus (WDV) to specific sites in the rice genome cleaved by the CRISPR/Cas9 system, and the homologous recombination repair efficiency reached 19.4%. However, the application of the HR pathway in crops is not as common as that of the NHEJ pathway, mainly because of the cell cycle dependence of the repair pathway, i.e., NHEJ can play a stable role in almost the entire cell cycle, while the HR pathway is only active in the S and G2 phases [14][18].
2.1. ZFNs
ZFNs are the fusion of artificially modified zinc-finger protein (ZFP), having specific binding activity, and Fok Ⅰ endonuclease, having non-specific cutting activity [17][21]. Several amino acid residues on the α helix of ZFP play a direct role in the recognition of the target site and can pair with bases adjacent to it. For example, the −1, +3, +6 locus on the α helix of Zif268 (a kind of ZFP) can directly recognize and bind to three adjacent bases on the target sequence [18][22] (Figure 1). In addition, the binding domain and cutting domain of the Fok Ⅰ endonuclease can be separated, and the non-specific cutting function of the Fok Ⅰ endonuclease can be obtained when the binding domain is removed [19][23]. Finally, under the action of Fok Ⅰ dimer, the target site will be cut, and DSBs will be generated [18][22].
ZFNs, as the first generation of gene editing technology, have been successfully applied in animals at first and have attracted wide attention [10][14]. Subsequently, ZFNs were applied in corn [20][24], rice [21][25], and other crops. However, there are inherent flaws in this system. For example, the inter-ZFP context effect, that is, it is difficult to achieve high efficiency by simply linking specific ZFPs together [10][22][14,26]. In addition, there are shortcomings such as unsuitability for multiple editing, high off-target efficiency, and high cytotoxicity, which are gradually being overcome by the later gene editing tools [10][22][14,26]. Therefore, its application in crops is not well developed.
2.2. TALENs
The structural difference between TALENs and ZFPs is that TALENs use the transcription-activator-like effector (TALE) as their binding domain, and the binding function mainly depends on highly variable amino acids at the 12th and 13th position on the TALE [11][23][15,27]. In combination with them, a single base can be specifically identified. The deciphered recognition method can meet the needs of arbitrary base recognition [11][24][15,28]. In this system, the non-specific cutting domain is still Fok Ⅰ endonuclease, and a pair of TALENs will form a Fok Ⅰ endonuclease dimer after binding to the target site, resulting in DSBs [25][29].
TALENs are more targeted, less cytotoxic, have no contextual effect such as the one in ZFPs, and can be assembled in a modular manner [26][27][30,31]. Currently, they have been successfully applied in rice [28][32], corn [29][33], wheat [30][34], and other crops. However, TALENs’ binding sites are restricted by guanine nucleotides and are not suitable for multiple editing or highly methylated sites [10][31][14,35]. But, highly methylated loci are very important in gene regulation research, and multiple editing is of great significance in studying the interaction between genes and improving editing efficiency, so the application of TALENs is limited to some extent.
2.3. CRISPR/Cas9 System
The widespread application of gene editing technology in crop improvement at present largely benefits from the emergence of the CRISPR/Cas9 system, which is derived from the immune system of bacteria and archaea [32][36]. The CRISPR/Cas9 system was synthesized by connecting single-guide RNA (sgRNA) and Cas9 [10][14]. The sgRNA can bind to the target site after specific modification [10][14]. The RuvC domain of Cas9 protein can cleave the DNA strand with protospacer-associated motif (PAM) sites, and the DNA strand complementary to sgRNA is cleaved by the HNH domain of Cas9 protein [12][16]. The cutting site is near the PAM site [12][16].
In contrast, when using the three gene editing systems for specific editing, ZFNs and TALENs need to modify at the protein level, while CRISPR/Cas9 system only needs to appropriately modify sgRNA at the RNA level for targeted modification, which makes engineering feasibility stronger. At the same time, the CRISPR/Cas9 system also has the advantages of being suitable for multiple edits and more efficient in generating insertions or deletions [22][26]. However, its off-target effect and restriction of targeting by PAM sites are its main defects [32][33][36,37]. Off-target effects tend to produce unexpected mutations that are often detrimental to the cell. In bacteria and archaea, PAM sites are used to distinguish their own sequences from foreign sequences and are necessary for recognition and cutting [32][36]. Nevertheless, as a gene editing tool, the dependence on PAM sites limits the system’s ability to target genes widely. Therefore, reduction of off-target effects and freedom from confinement to PAM sites can broaden the application range of CRISPR/Cas9. It has been reported that off-target effects can be effectively reduced through Cas9 modification, sgRNA modification, bioinformatics analysis, delivery mode optimization, and other methods [34][35][36][37][38][39][38,39,40,41,42,43]. The dependence of CRISPR/Cas9 on PAM sites can be reduced through two approaches: directed evolution and structural orientation [40][44]. On the premise of known enzyme structure, enzyme mutants are usually engineered using a structure-oriented approach.
With the advent of base editors (BEs) and prime editors (PEs), the CRISPR/Cas system has been further expanded. It can be edited accurately without DSBs. Cytosine base editors (CBEs), adenine base editors (ABEs), and glycosylase base editors (GBEs) have been developed successively in animal cells [41][42][43][44][45][45,46,47,48,49]. These BEs can realize the substitution of C–T, A–G, C–A, and C–G by selective artificial fusion with deaminase, glycosylase inhibitor, or glycosylase based on Cas9 nickase (nCas9) and sgRNA [41][42][43][44][45][45,46,47,48,49]. However, when these tools act directly on crops, they are extremely inefficient and cannot meet the needs of users. Hua et al. [46][50], based on the research of the David Liu team, developed a single base editor, ABE7-10, for plants by optimizing ABE deaminase. In addition, the introduction of A3A-PBE [47][51], PhieCBEs [48][52], pDuBE1 [49][53], and other base editors can greatly improve the efficiency and scope of application of plant base editing. PEs consist of reverse transcriptase (RT)-nCas9 and pegRNA (composed of sgRNA, reverse transcriptase template, and primer binding site (PBS)) [50][54]. The RT template was used as donor DNA, and RT-mediated reverse transcription was performed [44][50][48,54]. Thus, genes can be accurately knocked in or out while avoiding the inefficient HR repair pathway. In addition, it has the advantages of producing fewer by-products, the editable base substitution of all types, and little restriction by PAM sites [44][50][48,54]. These research results were initially realized in animal cells, so it is worth thinking about how to make full use of its powerful function in plants. Lin developed the plant prime editor (PPE) system by optimizing codons, promoters, and editing conditions, which introduced powerful functions into plants for the first time and laid the foundation for subsequent optimization.
Through these efforts, the development of gene editing technology has been deepened, providing an effective tool for accelerating crop domestication, enriching the crop gene pool, and improving crop yield and quality.
